Background: Olfactory ensheathing cells (OEC) are considered to be the most suitable cells for transplantation therapy in the central nervous system (CNS) because of their unique ability to help axonal regrowth and remyelination in the CNS. However, there are conflicting reports about the success rates with OEC. Aim: This study was undertaken to evaluate the therapeutic effect of OEC in rat models using different cell dosages. Material and Methods: OECs harvested from the olfactory mucosa of adult white Albino rats were cultured. Spinal cord injury (SCI) was inflicted at the lower thoracic segment in a control and test group of rats. Two weeks later, OECs were delivered in and around the injured spinal cord segment of the test group of the rats. The outcome in terms of locomotor recovery of limb muscles was assessed on a standard rating scale and by recording the motor-evoked potentials from the muscles during transcranial electrical stimulation. Finally, the animals were sacrificed to assess the structural repair by light microscopy. Statistical Analysis: Wilcoxon signed rank test and Mann-Whitney U-test were used to compare the data in the control and the test group of animals. A P value of <0.05 was considered significant. Results: The study showed a moderate but significant recovery of the injured rats after OEC transplantation (P=0.005). Conclusion: Transplantation of OECs along with olfactory nerve fibroblasts improved the motor recovery in rat models with SCI.

Spinal cord injury (SCI) is associated with significant neurological disability. The current management includes stabilization of the vertebral column and intensive rehabilitation depending on the level and extent of the injury. [1],[2] The scope of spontaneous recovery following complete SCI is limited due to a variety of reasons. Factors that hinder axonal regeneration following SCI include glial scar from astrocytes, inhibitory molecules, loss of oligodendrocytes that myelinate the axons and inability of the neurons to regenerate. Although many exciting experimental approaches have been developed to overcome these obstacles individually, an optimal therapeutic strategy for patients will require combinatorial treatments that address the extrinsic and intrinsic barriers to regeneration. [3],[4] Olfactory neurons retain the ability to regenerate throughout one's adult life. The olfactory ensheathing cells (OECs), a type of glial cells that ensheath the olfactory axons, facilitate the axonal regeneration by guiding the olfactory axons toward the central nervous system (CNS). Several studies highlight the potential therapeutic role of OECs in spinal cord repair. [5],[6] OECs reside in the lamina propria of the olfactory mucosa and in the olfactory bulb. Like the Schwann cells in the peripheral nervous system, the OECs surround the olfactory axons from the nasal mucosa to the olfactory bulb, a zone between peripheral and central nervous systems. The OEC has properties of Schwann cells in permitting axonal regeneration and it also behaves like astrocytes in residing and supporting the neurons within the CNS. These unique characteristics make OEC a cell of choice for CNS repair. [7] Furthermore, the OECs do not normally myelinate olfactory axons but, when transplanted into the spinal cord, they myelinate the regrowing axons in the region of injury, a property that is being exploited. [8],[9] However, reports show that the success rate for CNS repair with OEC is not consistent. This could be due to differences in cell dosages or follow-up period after transplantation. The aim of the present study was therefore to evaluate the therapeutic ability of OECs and the accompanying olfactory nerve fibroblasts (ONFs) to repair experimentally inflicted SCI in rat models using different cell dosages.

» Material and Methods

Experimental animals

Adult male and female Albino Wistar rats weighing 100-250 g were used for the study. The animals were procured from the institutional animal house facility and maintained as guided by the institutional animal ethical committee. They were fed with a normal diet as prescribed by the veterinarian of the animal house.

Collection of rat olfactory mucosa

The animal was anesthetized with an intraperitoneal injection of ketamine and xylazine (90:10 mg/kg body weight). The nasal septum and the covering mucosa were exposed through a mid-sagittal skin incision. The olfactory mucosa was identified by its posterior location and its yellow color. It was dissected out and processed for histological study and cell culture. Semi-thin sections of the olfactory mucosa were cut and stained with toluidine blue for the light microscopy study.

Culture of OECs and ONFs

The OECs and ONFs were harvested from the lamina propria of the olfactory mucosa, as described in the literature. [10] The olfactory mucosa was transported in cold culture medium (DMEM with Ham's F12 - 1:1 Gibco) to the cell culture lab. The olfactory epithelium was separated from the lamina propria using dispase II. The separated lamina propria was treated with collagenase type II, followed by 0.1% trypsin, and centrifuged. The cell pellets of OECs and ONFs were plated on a poly-L lysine-coated culture flask at a concentration of 5000-8000 cells/cm 2 . The culture was maintained at 37°C with 5% carbon dioxide in air at 95% humidity. Fresh medium was supplemented every second day by replacing half of the culture medium. Once the culture attained confluence, the cells were passaged.

Adult female Albino Wistar rats (150-250 g) were anesthetized with ketamine and xylazine (90:10 mg/kg) administered intraperitoneally. T10 laminectomy was completed through a 2 cm midline incision to expose the spinal cord. A custom-fabricated impactor device was developed so that a 10 g rod falls from a 20 cm height to produce a drop-weight injury. The injury was standardized by recording the fall and its retraction on an oscilloscope and calculating the force after transferring the data onto a computer [Figure 1]. The incision was closed with absorbable sutures. These rat models of SCI were grouped into the control group (n=10) and the test group (n=10). The control group of paraplegic rats received no cell transplantation while the test group received cell transplantation. Both groups received the same postoperative care, which consisted of twice-daily monitoring of general health, mobility, expression of the urinary bladder and bowel, administration of Ringer lactate, analgesics and antibiotics.

Figure 1: Graph shows the trajectory of the rod in the spinal cord impactor, when it is dropped and retracted to make a drop weight injury to the rat spinal cord. The graph shown is the position of the rod as detected using a displacement sensor

Cell transplantation was carried out in the test group of animals approximately 2 weeks following the injury. On the day of transplantation, few minutes before cell transplantation, the cultured OECs and ONFs were collected from the culture plate by trypsinisation and the enzymatic activity was stopped with fetal bovine serum. The cells were counted using a Neubauer counting chamber. The cells were washed with phosphate-buffered saline (PBS), centrifuged, pelleted and loaded into a sterile 25 μl Hamilton syringe (approximately 100,000 cells/μl of PBS). The Hamilton syringe was mounted onto an injection device with a 3D stabilizer for administration into the spinal cord. Rats were reanesthetized and the original injury site was reopened. Under a surgical microscope, the injured spinal cord segment was exposed and the cell suspensions were injected at multiple sites in and around the injury site. Cell doses ranging from 7 lakhs to 20 lakhs were administered [Table 1]. Following cell transplantation, the surgical wound was closed and routine postoperative care was given.

Table 1: Data of the test group (transplanted rats) and the control group showing time intervals, cell dosage, BBB score and amplitude of motor-evoked potentials

The BBB scale is an operationally defined 21-point scale designed to assess hind limb locomotor recovery after impact injury to the thoracic cord in rats. [11] Rats were placed in a floor area of 95 cm×95 cm. The bladder was emptied before testing. Limb movements of the animal were video graphed for BBB assessment. Open-field observations were made on all experimental rats. The BBB motor scores were evaluated independently by three investigators to minimize bias.

Transcranial electrical stimulation

The functional integrity of the spinal cord was evaluated by stimulating the motor cortex electrically across the scalp and recording the motor response from the lower limb muscles. A sample was also recorded from the upper limb muscles to assess the efficacy of the cortical stimulation. A custom-fabricated device was developed for transcranial stimulation of the motor cortex. The stimulating electrodes of this device were placed on the scalp overlying the motor cortex and the recording bipolar electrodes were inserted into the muscles of the anesthetized rats. Motor-evoked potentials from the gastrosoleus muscle of the lower limb and the triceps brachii of the upper limb were recorded in all rats before and after cell transplantation. The coinvestigators of the Bioengineering Department, who studied the transcranial motor-evoked potential studies, were blinded to the different groups of rats.

Histological study

After the above evaluations, the rats were anesthetized and transcardially perfused with 250 ml of PBS followed by 500 ml of 4% paraformaldehyde. The injured site of the spinal cord was reexposed and the injured segments along with the adjacent cranial and caudal segments were removed, from which 7-μ-thick longitudinal cryosections were cut and stained to detect the presence of p75 neurotrophin receptor cells. Similar spinal cord sections were made from the injured site of the control rats.

Statistical analysis

Wilcoxon signed rank test was used to compare the data in the test rats before and after cell transplantation. The Mann-Whitney U-test was performed to compare the data between the test and the control rats. A P-value of <0.05 was considered significant.

Statement of ethics

The Institutional Review Board and the Animal Ethics Committee approved all experimental protocols and procedures. All applicable institutional and Government regulations concerning the ethical use of animals were followed during the course of this research.

» Results

I. Rat olfactory mucosa

Histology

The olfactory epithelium was identified at the postero-superior corner of the rat nasal septum by its color, location and texture. The olfactory epithelium is thicker and has a yellowish appearance on the epithelial surface. The respiratory mucosa is thinner and is separated from the olfactory mucosa by a semicircular line. Toluidine blue staining studies of the nasal septum demonstrated the olfactory mucosa with olfactory nerve bundles in the lamina propria located on either side of the septum [Figure 2].

Figure 2: (a) Rat septum showing the position of the olfactory mucosa. Note the yellow color of the mucosa and the line of demarcation between the respiratory (RM) and the olfactory mucosa (OM). (b) Rat olfactory mucosa semithin section stained with toluidine blue. E, olfactory epithelium; S, nasal septum; NF, olfactory nerve fascicle within the lamina propria

Immunochemical staining of the cultured cells displayed two types of cell population, cells positive for neurotrophin p75 surface receptor identified as OECs and cells positive for fibronectin identified as ONFs [Figure 3]. Cells positive for p75 displayed a brown color as a DAB reaction product was present in these cells, whereas the cells nonreactive to the antibody were stained blue with the counter stain.

Figure 3: (a) p75 positive OEC in culture. Note the brown reaction product of DAB. The negative cells, stained with hematoxylin, are ONF. OEC and ONF form equal proportion of the cells related to the olfactory nerve in the olfactory mucosa. (b) Fibronectin positive ONF. Brown reaction product of DAB is seen in the cytoplasm of cells

There was no significant difference in the mean BBB score of the injured animals in the control group and in the test group before cell transplantation (P>0.05; [Table 2]). The mean BBB score of the test group of animals was significantly higher after cell transplantation than before transplantation (P<0.01) [Table 2]. The individual BBB score of all the injured animals improved after cell transplantation [Table 1]. Further, the mean BBB rating of the cell-treated group of animals was significantly higher than the control untreated group (P=0.0001; P<0.05) [Table 2], [Figure 4].

Figure 4: BBB score of the test group (n=10) before and after transplantation, and of the control rats (n=10)

Motor-evoked potentials recorded from the forelimb muscles of both the control group and the treated test group were normal. On the other hand, the amplitude of the motor-evoked potentials from the gastrocnemius muscles of the transplanted test group was significantly higher (P=0.004) than the control animals [Table 3], [Figure 5].

Figure 5: Motor evoked potential: (a) from control rat, (b) from transplanted rat. In both the graphs, upper curves were recorded from triceps brachii of fore limb and the lower curves were from gastrocnemius of hind limb. There is no electrical signal from lower limb muscles of the control group; however, a small, yet appreciable amplitude of action potential is seen in the gastrocnemius of cell- transplanted animal indicated by the arrow

The longitudinal section of the injured region of the spinal cord, stained with hematoxylin-eosin, appeared as a constricted thinned-out segment. Immunostaining of this injured segment of the transplanted cord showed brown-stained p75-positive cells, indicating the survival of the transplanted OECs in the spinal cord [Figure 6].

Figure 6: (a) Longitudinal section of cell transplanted spinal cord stained with H and E. The constricted area of the cord marked I- indicates the injured segment. (b) Immunostaining of the rat spinal cord for olfactory ensheathing cells after the transplantation. p75 positive cells (OEC) are stained brown indicating the presence of surviving transplanted OECs in the spinal cord

In the field of regenerative medicine, there is an urge for a novel cell-based therapeutic strategy that will allow for efficient treatment or even potential replacement of damaged organs. OECs and ONFs were used in our study in rat models of SCI based on prior reports of their role in CNS repair. [12],[13],[14],[15],[16],[17],[18] Neurogenesis occurs in the olfactory system throughout the life of adult mammals, facilitated by the OECs. It has been shown that transplanted OECs are capable of remyelinating the demyelinated axons in experimentally induced lesions of the animal spinal cord. [19] Transplantation of OECs enhanced axonal regeneration and improved recovery of locomotor function in experimental animals. [20] It has been shown to coexist in an astrocyte-rich environment fully integrating within a lesion. OECs can integrate with astrocytes in coculture as well without inducing astrocyte hypertrophy or upregulating chondroitin sulfate proteoglycan expression in them. [21],[22] On the basis of these encouraging reports in the literature, we initiated a study to evaluate the efficacy of various cell dosages of OEC transplantation in repairing the injured spinal cord in rat models.

The histological study of the rat olfactory mucosa and the immunocytochemical study of the cells cultured from the rat olfactory mucosa confirmed the presence of viable OECs and ONFs in the isolated rat olfactory mucosa. The close proximity between the processes of the OECs and the nerve fibers in the rat olfactory mucosa indicate that the OECs provide a structural support to the axons. Although the ONFs do not have direct contact with the axons, their location on the outer surface of the OECs suggest that the ONFs may be supporting the OECs.

The experimentally inflicted SCI was standardized by the use of the custom-fabricated impactor device. This ensured that the injury in the control and the test group of rats was comparable, a fact further corroborated by the absence of a significant difference between the BBB score of the control rats and the test rats before transplantation. This model was selected as it simulated the common mode of SCI and the cascade of cellular and molecular events causing secondary tissue damage, demyelination and apoptosis following trauma in patients. [23] Transplantation of the cultured cells into the injured spinal cord of the paraplegic rats significantly improved the BBB score when compared with the pretransplantation scores (P=0.002) and when compared with the control paraplegic rats that received no treatment (P=0.0001). Moreover, histological examination of the injured spinal sections of the transplanted group showed the presence of OEC cells alongside the typical pathological features of SCI. This demonstrated that some of the injected OEC cells survived and were in contact with the axons. The significantly larger motor-evoked potentials recorded in the gastrocnemius muscles of the cell-transplanted animals when compared with the control untreated injured animals (P=0.004) is further evidence that OEC and ONF transplantation has improved the motor recovery of the muscles below the level of injury.

OEC survive transplantation, integrate with the local environment, facilitate axonal growth and have the potential for auto-transplantation. [24] In the olfactory nerves, there is a close relationship between the OECs that ensheath the olfactory axons and the ONFs that support the OECs. The OEC along with the ONF have been observed to maintain a mechanically stable channel for the regenerating axon in the normal olfactory mucosa. [25],[26] In our study, the cell culture from the olfactory mucosa was not purified to separate out the OECs. Thus, the transplanted cells contained a mixture of OEC and ONF, both of which are considered to be essential for axonal regeneration. The transplanted OECs may induce recovery of function by stimulation of the preserved axons or remyelination of the spared demyelinated fibers. In addition to the possible direct effects of the transplanted OECs on the axons, the capacity of these cells to reconstruct an anatomical pathway facilitates axonal growth. [27]

Unlike previous reports, in our study, we administered different doses of cells and followed-up the effects for different periods of time by evaluating the BBB score and motor-evoked potential studies. The BBB score of the posttransplanted animals ranged from 1 to 17, with six of the 10 rats scoring more than 5. The corresponding BBB score in the control group as well as in the pretransplant group ranged from 0 to 1 [Figure 6]. Different cell dosages have been used by other investigators. [28],[29] In this study, it was planned to observe any dose-response relationship with increasing cell dosage. We administered cell dosages ranging from 7 to 19.8 lakhs. An improvement in the BBB motor score was observed following all dosages. Minimal improvement was seen with dosages ranging from 11 to 15 lakhs. An apparent dose-response relationship with incremental motor score was seen in four rats that received dosages higher than 16 lakhs. However, the maximum response was observed in the rat that received a dose of 9.6 lakhs. Considering the duration of follow-up after transplantation, five rats followed-up for more than 70 days showed a BBB score above 5. The rat that was followed-up to 264 days progressed to the highest BBB score of 17, suggesting that the duration after transplantation also has a role to play in the extent of recovery.

The results of our study demonstrate that allogenic transplantation of OECs in rats with SCI at the thoracic level improved the conduction of the spinal cord and the motor function of the lower limbs, suggesting recovery of the spinal cord from injury. Further studies involving tract tracing will confirm whether the recovery is due to axonal regeneration. The encouraging results of our study in rats suggest that OEC transplantation could be a logical approach to improve the outcome following SCI, the cure of which is still an imbroglio.